PROJECT SUMMARY/ABSTRACT The nuclear receptor ROR?t directs the differentiation of T cells that have critical roles in multiple autoimmune and inflammatory diseases. Pharmacologic targeting of ROR?t is hence being developed to treat diseases such as psoriasis, rheumatoid arthritis, and inflammatory bowel disease that are thought to be mediated in large part by Th17 cells and related cells that produce IL-17 cytokines. However, ROR?t has multiple other functions in diverse cell types, including mediating survival of thymocytes and regulating the development of lymphoid tissue inducer (LTi) cells, that guide the formation of secondary and tertiary lymphoid tissues, and of type 3 innate lymphoid cells (ILC3) that protect epithelial barriers. In addition, ROR?, a closely related isoform encoded by the same gene, has metabolic functions in multiple tissues, including liver and adipocytes. How ROR? and ROR?t function in different cell types to direct distinct transcriptional and functional programs is not understood. The transcriptional networks regulated by ROR?t and multiple other transcription factors have been studied in Th17 cells polarized in vitro with combinations of cytokines, but how ROR?/?t functions in Th17 cells and other cell types in vivo is not yet known. We have used a proteomics approach to characterize macromolecular complexes that govern ROR?/?t functions in different cell types and identified DDX5, a DEAD- box RNA helicase, as associated with ROR?t in Th17 cells. In mice deficient for DDX5 in T cells, expression of multiple ROR?t target genes is attenuated and the animals are resistant to Th17 cell-mediated inflammatory disease. We also found the long noncoding (lnc) RNA Rmrp associated with DDX5 and with ROR?t, both in Th17 cells and when all three components were synthesized in vitro. Mutations in Rmrp result in cartilage-hair hypoplasia (CHH), a recessive human genetic disease characterized by abnormal bone growth, immune deficiency and neuronal dysplasia in the intestine. Expression of wild-type, but not a CHH mutant, Rmrp promoted Th17 cell differentiation in a DDX5-dependent manner, indicating that this is a rare lncRNA that functions in trans. Our results suggest that Rmrp binding to DDX5 promotes the interaction of DDX5 with ROR?t and coactivation of ROR?t target genes. We propose in Specific Aim 1 to identify gene targets whose expression is dependent on DDX5, Rmrp, and ROR?t, using genome-wide transcriptomics, chromatin accessibility, and chromatin occupancy studies with in vitro polarized or in vivo generated Th17 cells from wild type and mutant animals. In Specific Aim 2, we will determine, using biochemical approaches, how these molecules interact with each other and whether this interaction is restricted to Th17 cells or is present in other ROR?/?t-dependent cells. In Specific Aim 3, we will characterize the influence of the interactions in Th17 cell- mediated inflammation, ILC3-dependent barrier protection, and development of thymocytes and lymphoid tissues. Our results will provide a better understanding of the functions of ROR?t, which may facilitate design of more specific therapeutics for autoimmune disease and for immune deficiency in CHH patients.